This is a collaboration with Y-X Wang (SBL) to optimize expression of continuous repeat unit CR567 of Lipoprotein Receptor Related Protein (LRP)in order to study its binding kinetics with RAP (Receptor Related Protein)and to determine the structure of the C567-RAP complex. LRP is a transmembrane receptor protein that binds with more than 30 ligands in vivo, making it an important receptor in many cellular processes. LRP has many structural domains;domain II contains several continuous repeat (CR) units including CR567. Previous studies have shown that the CR567 units are important for RAP binding, although the role of the specific CR units in RAP binding is still poorly understood. The signaling pathway of LRP is tightly controlled by a number of extracellular and intracellular regulating proteins, including chaperonins and members of the Dickkopf (DKK) family. These proteins down-regulate Wnt-B-catenin pathway as tumor suppressors. LRP6 has been shown to be an oncogenic receptor. This pathway has high potential for molecular targeting. The ligand binding sites of CR567 are cysteine-rich complement type repeats, making expression and purification with proper folding difficult, a major hurdle to biophysical and structural studies. Other laboratories have expressed CR56 as a ubiquitin fusion protein, which is the approach we are using to express CR567. We have cloned the gene encoding CR567 into pET SUMO cloning vector also containing throredoxin, and expressed protein using the origami E. coli strain. The resultant protein can be separated from aggregates, is clear when labeled with N15, and gives a better resolution NMR spectrum than has been obtained previously. We are working on binding assays using SPR,intrinsic fluorescence, and ITC. This work will be continued in Dr. Wangs laboratory after closure of my section at the end of FY09.